Methods for Detecting and Analyzing N-Glycolynlneuraminic Acid (Neu5Gc) in Biological Materials

ABSTRACT

The present application is in the field of sialic acid chemistry, metabolism and antigenicity. More particularly, the present invention relates to the detection and analysis of the non-human sialic acid, N-glycolylneuraminic acid (Neu5Gc) in bio-logical materials, such as food and clinical specimens. Such detection and analysis is facilitated by the use of Neu5Gc specific antibodies. The present invention also relates to the detection of anti Neu5Gc antibodies in clinical samples, as well as the production of anti-Neu5Gc specific antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application60/487,533, which was filed on Jul. 15, 2003.

GOVERNMENT INTEREST

This invention was made with government support from the NationalInstitutes of Health under grant number R01GM32373.

TECHNICAL FIELD

The present application is in the field of sialic acid chemistry,metabolism and antigenicity. More particularly, the present inventionrelates to the detection and analysis of the non-human sialic acid,N-glycolylneuraminic Acid (Neu5Gc) in biological materials, such as foodand clinical specimens. The present invention also relates to thedetection of Neu5Gc antibodies in clinical samples, as well as theproduction of anti-Neu5Gc-specific antibodies.

BACKGROUND

All cells are covered with a dense and complex array of sugar chains.Sialic acids (Sias) are a family of nine-carbon sugars that aretypically present at the outermost units of these sugar chains. Byvirtue of their terminal position, sialic acids act as binding sites formany exogenous and endogenous receptors such as the Influenza virusesand the Siglec family of endogenous proteins. Such sugars are thususeful drug targets for the prevention and treatment of infection. Theyare also involved in various biological and pathological processes suchas neuronal plasticity and cancer metastasis. In many of theseinstances, the precise structures of the sialic acid and the residues itis attached to play critical roles. Thus, studying sialic acid functionsis of great biological importance, and antibodies specific for sialicacids are valuable tools in elucidating their biological functions. Morespecifically, these antibodies would be useful to screen normal humantissues for traces of Neu5Gc, which may be incorporated from certaindietary sources (red meat and diary products), and may also beassociated with certain disease states, such as cancer and heartdisease.

Sialic acids are typically found as the outermost-units on the mammaliancellular glycocalyx, and on secreted glycoproteins. (Gottschalk, A.(1960), The Chemistry and Biology of Sialic Acids, University Press,Cambridge, U.K.; Rosenberg, A. and Schengrund, C. (1976), Bilogy ofSialic Acids, Plenum Press, New York, N.Y.; Schauer, R. (1982) Adv.Carbohydr. Chem. Biochem. 40:131-234; and Angata, T. and Varki, A.(2002) Chem. Rev. 102:439-470.) The most common Sias areN-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc).Cellular Neu5Gc is generated by hydroxylation of the sugar nucleotidedonor CMP-Neu-5Ac to CMP-Neu-5Gc, catalyzed by CMP-Neu5Ac hydroxylase(CMAH). (Shawl L. and Schauer, R. (1988) Biol. Chem. Hoppe-Seyler369:477-486; Kozutsumi, Y., Kawano, T., Yamakawa, T. and Suzuki, A.(1990) J. Biochem. (Tokyo) 108: 704-706; and Muchmore, E. A., Milewski,M., Varki, A., and Diaz, S. (1989) J. Biol. Chem. 264: 20216-20223.)

Although Neu5Gc is a major Sia in most mammals (including our closestevolutionary relatives, the great apes; Muchmore, E. A., Diaz, S. andVarki, A. (1998) Am. J. Phys. Anthropol. 107: 187-198), it is thought tobe absent in healthy humans (Gottschalk, et. al.; Rosenberg, et al.; andSchauer, supra). Indeed, humans generate immune responses againstintravenously administered molecules carrying Neu5Gc, e.g. the “serumsickness” reaction to equine anti-thymocyte globulin therapy (Higashi,H., Naiki, M., Matuo, S. and Okouchi, K. (1977), Biochem. Biophys. Res.Comm., 79: 388-395; and Merrick, J. M., Zadarlik, K. and Milgrom, F.(1978), Int. Arch. Allergy Appl. Immunol. 57: 477-480). These findingsare explained by a human-specific inactivating mutation in the CMAH genethat occurred 2.5-3 million years ago (Chou, H. H., Takematsu, H., Diaz,S., Iber, J., Nickerson, E., Wright, K. L., Muchmore, E. A., Nelson, D.L., Warren, S. T. and Varki, A. (1998), Proc. Natl. Acad. Sci. USA95:11751-11756; Irie, A., Koyama, S., Kozutsumi, Y., Kawasaki, T. andSuzuki, A. (1998), J. Biol. Chem. 273:15866-15871; Varki, A. (2002),Yearbook Phys. Anthropol. 44:54-69; and Chou, H. H., Hayakawa, T., Diaz,S., Krings, M., Indriati, E., Leakey, M., Paabo, S., Satta, Y.,Takahata, N. and Varki, A. (2002), Proc. Natl. Acad. Sci. USA99:11736-11741).

Despite no known alternate pathway for Ne45Gc synthesis in humans,antibodies have been used to claim its presence in some human cancersand in human fetal meconium. (Hirabayashi, Y., Kasakura, H., Matsumoto,M., Higashi, H., Kato, S., Kasai, N. and Naiki, M. (1987), Japan J.Cancer Res. 78:251-260; Higashi, H., Hirabayashi, Y., Fukui, Y., Naiki,M., Matsumoto, M., Ueda, S. and Kato, S. (1985), Cancer Res.45:3796-3802; Miyoshi, I., Higashi, H., Hirabayashi, Y., Kato, S. andNaiki, M. (1986), Mol. Immunol. 23: 631-638; Marquina, G., Waki, H.,Fernandez, L. E., Kon, K., Carr, A., Valiente, O., Perez, R. and Ando,S. (1996), Cancer Res. 56: 5165-5171; Devine, P. L., Clark, B. A.,Birrell, G. W., Layton, G. T., Ward, B. G., Alewood, P. F. & McKenzie,I. F. C. (1991), Cancer Res. 51:5826-5836; and Kawachi, S., Saida, T.,Uhara, H., Uemura, K., Taketomi, T. and Kano, K. (1988), Int. Arch.Allergy Appl. Immunol. 85:381-383.) However, the specificity of thepolyclonal antibodies used was not well defined.

One study using mAbs failed to detect Neu5Gc in human tumors and tissues(Furukawa, K., Yamaguchi, H., Oettgen, H. F., Old, L. J. and Lloyd, K.O. (1988), J. Biol. Chem. 263:18507-18512). However, these mAbs werespecific for Neu5Gc only in the context of underlying structural motifs.Another mAb thought to be specific for Neu5Gc cross-reacts with somesulfated glycolipids (Vázquez, A. M., Alfonso, M., Lanne, B., Karlsson,K. A., Carr, A., Barroso, O., Fernández, L. E., Rengifo, E., Lanio, M.E., Alvarez, C., Zeuthen, J. and Pérez, R. (1995), Hybridoma14:551-556). Meanwhile, some reports claim chemical proof for Neu5Gc inhuman tumors Marquina, et al., Devine, et al., supra, and Kawai T.,Kato, A., Higashi, H., Kato, S. and Naiki, M. (1991), Cancer Res.51:1242-1246). Overall, prior data is inconclusive about the frequencyand distribution of Neu5Gc expression in tumors.

Human biosynthetic pathways could theoretically allow exogenous Neu5Gcto be metabolically incorporated (Varki A., et al., supra, and Oetke,C., Hinderlich, S, Brossmer, R., Reutter, W., Pawlita, M. and Keppler,O. T. (2001), Eur. J. Biochem. 268:4553-4561). Indeed, human cellscultured in fetal calf serum express cell surface Neu5Gc in smallamounts (Muchmore, et al., and Furukawa, et al., supra). However, it isnot known if this represents passive adsorption of serum glycoconjugatesor metabolic incorporation. Although earlier studies claimed the absenceof Neu5Gc from normal human tissues, a small HPLC peak was noted at theelution time of Neu5Gc in extracts from human organs (Muchmore, et al.,supra). More recent studies as discussed elsewhere herein haveestablished the pathway for uptake and incorporation of Neu5Gc by humancells.

Anti-Neu5Gc antibody levels in human sera have also been reported,primarily in patients with various diseases. The presence of suchantibodies in normal individuals was considered to be rare. However,assays used in all previous studies lacked absolute specificity forNeu5Gc and also suffered from very high background signals. Thus, theylacked the sensitivity to detect low levels of anti-Neu5Gc antibodiesand failed to be certain about the specificity of the reactingantibodies. For example, one study used high molecular weightglycoproteins (HMWG) extracted from bovine erythrocytes as a targetwithout a true negative control, and arbitrarily defined a positiveresult as an absorbance value of more than 0.5. Thus, only sera withvery high reactivity, such as those of patients with certain diseasestates, would have been identified as positive. Another study that alsoutilized HMWG as the target, without a true negative control, defined apositive result as the value obtained after subtracting the resultsobtained using non-coated wells from results obtained using HMWG coatedwells. The propensity for serum proteins to bind non-specifically tonon-coated wells would have also given a high background in this assay,again making it difficult to identify weakly positive-samples. In bothassays the specificity of the reacting antibodies would also not beclearly defined, since cross-reactivity with other bovine moleculescould occur.

Accordingly, the present invention relates to detection and analysis ofNeu5Gc content of biological materials, such as food and human clinicalsamples. It also relates to the study of induced and natural antibodiesthat are specific for Neu5Gc, and their use in the study of Neu5Gc inthe laboratory, as well as the detection of Neu5Gc antibodies inclinical specimens. The invention also relates to general methods forselecting anti-sialic acid specific antibodies.

SUMMARY OF THE INVENTION

The non-human sialic acid Neu5Gc is found in various food and can betaken up and incorporated into human tissues. This can be accomplishedby a human antibody response to Neu5Gc. The presence of the antigen andthe antibody in the same person could facilitate the occurrence of avariety of diseases. This invention relates to the detection andquantitation of Neu5Gc in food and clinical samples, as well asmeasurement of anti-Neu5Gc antibodies in order to make prognosticstatements about the risk of various disease and to facilitate dietaryrecommendations. The invention also includes methods for specificproduction and detection of sialic acid-specific antibodies.

In one embodiment, the present invention is a method of determining %N-glycolylneuraminic acid (Neu5Gc) of a biological material comprisingthe steps of: (a) measuring the amount of N-acetylneuraminic acid(Neu5Ac) present in the material per given weight; (b) measuring theamount of Neu5Gc present in the material per given weight; and (c)calculating the % Neu5Gc of the sample, wherein % Neu5Gc is determinedusing the formula: % Neu5Gc=[(Neu5Gc)/(Neu5Ac+Neu5Gc)]×100. Thebiological material can be virtually any organic material suspected ofcontaining Neu5Gc, such as a food sample (for example, red meat or adairy product) or a clinical sample. The clinical sample may be from anyanimal source, such as a human. Human samples can be from any body fluidor tissue, such as urine, saliva, soft or hard tissue, urine, etc.

The Neu5Gc content of food is particularly important. As such the Neu5Gccontent of food can be calculated on a “weight per serving” basis bymultiplying the Neu5Gc content per weight by the serving size.

When the % Neu5Gc is measured in a clinical sample, it may also beimportant to monitor changes in the % Neu5Gc over time, especially inresponse to dietary changes.

In another embodiment, the invention is a method of purifying sialicacid-specific antibodies, comprising the steps of: (a) preparingantibodies to sialic acids; (b) contacting the antibodies from step (a)with a first solid phase to which a sialic acid having a side chain hasbeen attached; and (c) contacting the antibodies that bound to the firstsolid phase with a second solid phase to which the sialic acid withoutthe side chain has been attached. The sialic acid can be any member ofthe sialic acid family. Examples include N-acetylneuraminic acid,N-glycolylneuraminic acid, Ketodeoxynonulosonate (KDN)N-propanoylneuraminic acid, N-butanoylneuraminic acid,N-pentanoylneuraminic acid, N-hexanoylneuraminic acid,N-heptanoylneuraminic acid, N-oxooctanoylneuraminic acid,N-levulinolylneuraminic acid, N-homolevulinoylneuraminic acid,N-oxohexanoylneuraminic acid, N-oxoheptanoylneuraminic acid, andN-oxooctanoylneuraminic acid, as well as natural and syntheticderivatives of the foregoing sialic acids.

Although any method may be used to remove the sialic acid side chaineither before or after attachment to the second solid phase, the use ofperiodate is preferred.

The present invention also includes a method of detecting anti-Neu5Gcspecific antibodies in a biological material comprising the steps of:(a) determining the amount of anti-Neu5Ac antibodies present in thematerial; (b) determining the amount of anti-Neu5Gc antibodies presentin the material; and (c) subtracting the amount of anti-Neu5Acantibodies from the amount of anti-Neu5Gc antibodies to determine theamount of anti-Neu5Gc specific antibodies. The same types of biologicalmaterials described above can be assayed for the presence of anti-Neu5Gcantibodies.

In yet another embodiment, the present invention relates to acomposition comprising an affinity purified antibody specific forbinding N-glycolylneuraminic acid (Neu5Gc).

The present invention additionally includes a method for purification ofantibodies specific to sialic acid groups comprising: (a) exposing anantibody containing solution to a first immobilized target that has avery low density of the sialic acid under conditions to allow antibodybinding; (b) challenging the bound antibodies with a second immobilizedphase comprising the first immobilized phase treated with mild sodiumperiodate; and collecting the antibodies that do not bind to the secondimmobilized phase.

It has recently been suggested that Neu5Gc may play a role in diseases,such as cancer and heart disease. Accordingly, the present inventionalso relates to a method for the diagnosis of disease comprising: (a)collecting a serum or tissue sample from an individual; (b) analyzingthe sample for an elevated level of Neu5Gc specific antibodies in serum;and (c) correlating an increased level of antibody with the presence ofdisease.

Other aspects of the present invention are described throughout thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Affinity-purification of a monospecific Anti-Neu5Gc antibody.The total IgY pool from eggs of chickens immunized with gangliosideG_(M3)(Neu5Gc) was pre-cleared over a column of immobilized bovineasialofetuin (fetuin with sialic acids removed, not shown). Theflow-through fraction was passed over a column of immobilized bovinefetuin (Panel A) with a very low density of bound Neu5Gc (2.7% of totalSias). This allowed only the highest affinity antibodies to bind. Thesewere eluted with mild acid and dialyzed against buffer. The flow-throughfraction was repeatedly reapplied, until no more antibodies bound. Thepooled bound fractions were then reapplied to a similar column ofimmobilized fetuin (Panel B), which differed only in that it had beenpretreated with mild periodate oxidation to selectively truncate theside chain of Sias. Repeated rounds of this process yielded a pool ofantibodies that had originally bound to the unmodified fetuin column,and continued to bind to the modified column.

The starting IgY fraction showed three times higher reactivity againstbovine serum glycoproteins (which carry both Neu5Ac and Neu5Gc), thanagainst human serum glycoproteins (which carry only Neu5Ac). Thetwo-step affinity purification process enriched for a subset of the highaffinity antibodies against Neu5Gc. Non-specific reactivity of theresulting antibody to human serum was reduced to background levels (notshown).

FIG. 2. Characterization of specificity of the anti-Neu5Gc antibody.ELISA against Neu5Ac-LacNAc-PAA or Neu5Gc-LacNAc-PAA. Data show the meanof three determinations.

FIG. 3. Mass spectrometry detection of urinary excretion and salivarymucin incorporation of ingested Neu5Gc. Sias were purified from theurine (A) and salivary mucins (B) of the three subjects before Neu5Gcingestion and at various subsequent time points, and analyzed byDMB-derivatization, HPLC and MS. The sum of the intensities of ions atm/z 424 and m/z 442 at each time point is plotted for each subject.

FIG. 4. ELISA detection of Anti-Neu5Gc antibodies in normal human seraand demonstration of specificity. A. Results are plotted as meanbackground values with PAA-Neu5Ac subtracted from the signal withPAA-Neu5Gc. The mean value for all positive sera in each sub-class isrepresented by the horizontal bar. B. Two of the human sera identifiedas medium or high positive for anti-Neu5Gc IgG antibodies to the targetNeu5Gc-PAA were tested in the same ELISA assay with dilutions ofchimpanzee serum added to inhibit binding. A human serum withundetectable anti-Neu5Gc antibodies was used as a control.

FIG. 5. Comparison of levels of anti-Neu5Gc and anti-α-Gal antibodies innormal human sera. Serum samples were collected several months laterfrom the same individuals studied in FIG. 4A. Levels of anti-Neu5Gcantibodies were studied identically, in comparison with parallel wellscoated with Galα1-3Galβ1-4GlcNAc-PAA, instead of Neu5Gc-PAA. The finalreadout of values was thus directly comparable between the two probes,after subtraction of the background readings with Neu5Ac-PAA. IgG levelswere quantified as described.

FIG. 6. Mass spectrometric detection of Neu5Gc in normal human kidneyglycopeptides. Sias were released and purified from human kidney tissue,derivatized with DMB and fractionated by HPLC. (A) Mass spectrometryanalysis showing ions at m/z 442 and 424, consistent with the molecularion of DMB-derivatized Neu5Gc and its dehydrated form. (B) SelectiveMS/MS on the m/z 442 ion gives a fragment ion of m/z 424, confirmingthat the original m/z 442 ion was DMB-derivatized Neu5Gc.

FIG. 7. Proposed pathways for uptake and incorporation of free Neu5Gcinto human cells. This model is based on the literature and studiesdescribed herein. Thickness of the arrows suggests the relativeimportance of various pathways in delivering Neu5Gc into the cell.

DESCRIPTION OF THE INVENTION

The present application is in the field of sialic acid chemistry,metabolism and antigenicity. More particularly, the present inventionrelates to the detection and analysis of the non-human sialicacid,_N-glycolylneuraminic Acid (Neu5Gc) in biological materials, suchas food and clinical samples. For example, the analysis of Neu5Gccontent in food provides useful dietary information. Additionally, theanalysis of Neu5Gc and antibodies thereto in clinical specimens isuseful in the prevention, prognostication and diagnosis of disease.

Preparation of Sialic Acid Specific Antibodies

a. Sialic Acids

Sialic acids are 9-carbon monosaccharides that have the generalstructure of N-acetylneuraminic acid (Neu5Ac), which is the most commonnatural form of sialic acid found in humans. Sialic acids have acarboxylic acid group and a hydroxy group attached to carbon 2, which isthe anomeric position of the molecule. The sialic acids differ accordingto the side chain attached to carbon 5 (the “side chain”, as usedherein), which is an N-acyl group in the Neu5Ac molecule. Many non-humannaturally occurring sialic acids are known, such as Neu5Gc, which has aCH₂OHCONH— side chain, instead of the CH₃CONH— side chain of Neu5Ac.Another exemplary sialic acid is Ketodeoxynonulosonate (KDN). Othersynthetic sialic acids have also been produced that, like Neu5Ac, can beincorporated into sialoglycoconjugates to varying degrees. Theseinclude, for example, N-propanoylneuraminic acid, N-butanoylneuraminicacid, N-pentanoylneuraminic acid, N-hexanoylneuraminic acid,N-heptanoylneuraminic acid, N-oxooctanoylneuraminic acid,N-levulinolylneuraminic acid, N-homolevulinoylneuraminic acid,N-oxohexanoylneuraminic acid, N-oxoheptanoylneuraminic acid, andN-oxooctanoylneuraminic acid. As used herein, reference to a particularsialic acid intends the naturally occurring sialic acid, the syntheticsialic acid, as well as any derivatives thereof.

b. Side Chain Removal

The sialic acid side chain can be removed by any oxidative chemicalreaction, but is preferably removed with periodate.

c. Production of Antibodies

Monoclonal and polyclonal antibodies to sialic acids can be producedusing known means. For example, chickens are known to beimmunoresponsive to Neu5Gc, and anti-Neu5Gc IgY antibodies can be raisedusing the methods described in the Examples.

d. Purification of Antibodies

The production of sialic acid-specific antibodies involves a two-stepprocedure. In the first step, antibodies produced against sialic acids(either polyclonal or monoclonal) are contacted with a first solid phaseto which the sialic acid of interest is attached. The bound antibodiesare then eluted from the solid phase and thereafter challenged with asecond solid phase to which the same sialic acid of interest without theside chain is attached (i.e. the “truncated form”). Antibodies that bindto the first solid phase but not the second solid phase are considered“specific” for the sialic acid of interest, since they do not bind tothe truncated form of the sialic acid.

Any solid phase to which sialic acids (or sialic acid-containingmolecules) can be attached is suitable for use in the method of thepresent invention. Such solid phases and means for attachment are wellknown to those of skill in the art. For example, various types ofcellulose or agarose columns or ELISA plates are readily available.

Elution of bound antibodies after binding to either solid phase can beeasily accomplished by washing the solid phase with the manufacturer'srecommended elution buffer, such as low pH.

In addition to being useful for purifying anti-sialic acid specificantibodies, this two step approach can be used to test the specificityof monoclonal antibodies raised against sialic acids, and/or screen fortheir presence in clinical samples.

Uses of Anti-Sialic Acid Specific Antibodies

The antibodies prepared as described above are useful for analyzing thesialic acid content of any biological materials. The sample to beanalyzed using the methods of the present invention may be from anysource that is suspected of containing the sialic acid of interest. Inthe research laboratory, the sample may be derived from food, animalmaterial, plant material, environmentally derived samples, etc., thesialic acid content of which is of interest to the investigator. In theclinical laboratory, the sample may similarly be derived from any food,animal material, human clinical sample, plant material, ingestedsubstance, bodily fluid, and the like, that is associated with aclinical subject, and suspected of containing the sialic acid ofinterest. In particular, human serum or other bodily fluids may beinvestigated to detect the presence of sialic acids and their relativeconcentrations as an indication of a dietary condition or disease state.

Testing for Neu5Gc in Food

Neu5Gc is known to be prevalent in food sources of mammalian origin, andin particular “red meat” and dairy products. Since Neu5Gc is known toaccumulate in human tissues when the subject ingests food substancescontaining Neu5Gc, it is important to determine the Neu5Gc content infood and to correlate intake with clinical results to assess thepathological significance of Neu5Gc content in clinical samples fromhuman subjects.

The absolute amount of Neu5Gc in foods per serving is important, butperhaps more importantly is assessing the value of % Neu5Gc, which isthe amount of Neu5Gc divided by the amount of Neu5Gc+all other sialicacids. Since Neu5Gc is predominant, the % Neu5Gc can be determined bydividing the amount of Neu5Gc by the amount of Neu5Gc+Neu5Gc.Correlating the % Neu5Gc in foods ingested by a human subject allows forthe accurate assessment of the total “body burden”.

The Neu5Gc content can be expressed in terms of absolute % Neu5Gc, sinceNeu5Gc is generally very low in content in vegetables, fish and poultry.The Neu5Gc “load” in foods can be expressed as the total dailyrecommended serving, multiplied by the Neu5Gc content. For example, asshown below in Table 2, butter has 3% Neu5Gc. Butter has a total of 40μg/g of sialic acids, which equates to 1.2 μg/g of Neu5Gc (40×0.03=1.2).If you multiply this amount by the total recommended daily allowance of15 grams of butter, the total Neu5Gc load of a daily serving of butteris 18 μg.

In addition, the Neu5Gc content of food on a “per serving” basis mayprove to be useful labeling information for foods. As shown in Table 2,this value is easily calculated by multiplying the Neu5Gc content by theserving size. “Weight per serving” values are found on virtually everyfood label and are readily available from a variety of different (e.g.government) sources.

The total body uptake of Neu5Gc from a given food source may not bedirectly correlated to its absolute Neu5Gc content because the extent towhich the food source is digested and Neu5Gc is released for adsorptionmight vary. The uptake of Neu5Gc from specific foods could also be usedto generate a Neu5Gc index.

A “Neu5Gc index” can be used in much the same way as the glycemic index,as a numerical system of measuring how fast a given food source triggersa rise in circulating Neu5Gc. The higher the number, the greater thepossibility of incorporation into body cells and tissue. A low “Neu5Gcindex” food will cause a small rise, while a high index food willtrigger a dramatic spike.

Testing for Neu5Gc in Clinical Specimens

The presence of Neu5Gc in human tissues may play an important role inthe pathology of diseases, because of the simultaneous presence ofanti-Neu5Gc antibodies circulating in the same person. Thus, Neu5Gc mayplay a role in heart disease, cancer, and other conditions. Also, sinceNeu5Gc elicits an antibody response in humans, in extreme cases, it maycause “serum sickness”.

In order to assess the clinical significance of Neu5Gc content inclinical specimens, it is desirable to measure not only the absolutevalue of Neu5Gc, but also the % Neu5Gc as described above and in theExamples.

Detection of Anti-Neu5Gc Antibodies

As discussed above, human exposure to Neu5Gc elicits an antibodyresponse. Since Neu5Gc is implicated in disease, the presence of highlevels of Neu5Gc antibodies may also be associated with disease. Asdescribed in the Examples, Neu5Gc antibodies are detected using routineantibody screening assays. Also as described, reactivity of human serawith Neu5Ac is measured as a control, and relative reactivity isdetermined.

EXAMPLES Example 1 Preparation and Characterization of ChickenAnti-Neu5Gc Antibodies

Generation of a Monospecific Chicken Polyclonal Antibody againstNeu5Gc—Lipids were extracted from horse erythrocytes (Rosenberg, et al.,supra), suspended in water, dialyzed against water, and fractionated onDEAE Sephadex A-25 (Schauer, et al., supra). Eluted fractions containingG_(M3)(Neu5Gc) were detected by HPLC, concentrated by evaporation,dialyzed against water and taken to dryness. Chickens were immunized andboosted with 1 mg each of G_(M3)(Neu5Gc) (Angata, et al., supra), exceptthat BSA was not methylated. Other Neu5Gc-containing antigens could alsobe used. Egg yolks were collected, a total IgY fraction obtained by 40%ammonium sulfate precipitation, and dialyzed repeatedly against PBS, pH7.4. Antibody titer was monitored by horse erythrocyte hemagglutination,and the pool stored at −20° C.

Columns for Affinity Chromatography—The column for pre-clearing had 81.2mg of bovine asialofetuin (Sigma, St. Louis, Mo.) coupled to 5 mlAffi-Gel 15 beads (BioRad, Richmond, Calif.). The first affinity columncontained 23.8 mg native (fully sialylated) fetuin (Sigma) coupled to 5ml of Affi-Gel 15. A modified fetuin-column with truncated Sia sidechains was prepared by treating 5 ml of an identical column with 25 mlof 2 mM sodium periodate in PBS, pH 7.4 for 30 min, rotating at 4° C. inthe dark. Ethylene glycol (250 μl of 5M) was added for 5 min at 4° C. toconsume excess periodate. After washing with 4×5 ml PBS, pH 7.4 and 4×5ml 50 mM sodium acetate buffer, pH 5.5, 20 ml of 20 mM acetic hydrazidein 50 mM sodium acetate buffer, pH 5.5 was added to block reactivealdehydes generated by periodate cleavage of the Sia side chain. After 2hrs in the dark at RT with gentle shaking, the beads were washed with2×20 ml of PBS and resuspended in 0.01% sodium azide/PBS until use.Completeness of side chain truncation was confirmed by release of Siasusing mild acid (2M acetic acid at 80° C. for 3 hrs), derivatizationwith 1,2-diamino-4,5-methylene-dioxybenzene (DMB) (Sigma) and HPLCanalysis (Shaw, et al., supra).

Affinitv Purification of Chicken anti-Neu5Gc antibody—The IgY fractionwas thawed, clarified at 100,000 g for 30 min, and 1 ml of supernatantpre-cleared by passage over the asialofetuin column. The flow-throughfraction was pooled with column washes of 2×5 ml PBS, pH 7.4, and passedover an affinity column containing native (sialylated) fetuin (see FIG.4, panel A). The flow-through and washes (2×5 ml PBS, pH 7.4) werecollected, and Neu5Gc-specific antibodies recovered by elution with 2×5ml 0.1M citric acid, pH 3.0, into a vial containing 2.4 ml of 1M Tris,pH 11. Eluted fractions were monitored by ELISA againstNeu5Gc-containing bovine serum (see below). The flow-through fractionwas reloaded onto the same fetuin column, washed and eluted again. Thisprocedure was repeated until there was no further binding of antibodies.

Removal of side-chain dependent anti-Neu5Gc antibodies—The pooledfetuin-binding eluate fractions were concentrated to 3 ml using aMillipore Ultrafree-15 centrifugal filter device with Biomax-30Kmembrane (Millipore, Bedford, Mass.), and then passed over the column offetuin with truncated Sia side chains (FIG. 1, panel B). Theflow-through fraction was collected along with washes of 2×5 ml PBS, pH7.4. The antibodies that continued to bind to this modified column werethen eluted with 2×5 ml 0.1M citric acid, pH 3.0, into a vial containing2.4 ml of 1M Tris, pH 11. The column was then re-equilibrated in PBS,and the above procedure was repeated with the flow-through fractionuntil there was no further decrease in reactivity to bovine serum. Thecombined eluates from all of the runs were concentrated to 3 ml using aMillipore Ultrafree-15 centrifugal filter device with Biomax-30Kmembrane.

Monitoring the Purification and Binding Specificity of the anti-Neu5GcAntibody—Microtiter plate (Costar, Corning, N.Y.) wells were coatedeither with bovine serum (containing both Neu5Ac and Neu5Gc), or humanserum (containing only Neu5Ac) at 115 μg protein/well, in 50 mM sodiumcarbonate-bicarbonate buffer, pH 9.5, at 4° C. overnight. Wells werewashed with Tris buffered saline (TBS), pH7.5, and subsequently blockedwith TBS+0.1% Tween (TBST) for 1 hour at RT. The wells were again washed5× with TBS, and incubated with serial dilutions of the antibody in TBSTfor 2 h at RT, washed 5× again with TBS, and then incubated with adonkey anti-chicken IgY antibody conjugated with horse radish peroxidase(Jackson ImmunoResearch, West Grove, Pa.) at a 1:10,000 dilution in TBSTfor 1 hour at RT. After 5 washes in TBS, antibody binding was measuredusing 2.75 mM O-phenylene-diamine (OPD) (Sigma,) and 0.075% hydrogenperoxide in citrate phosphate buffer, pH 5.5; quenching with 40 μl of 4Msulfuric acid, and reading absorbance at 492 nm with a microplatespectrophotometer (Molecular Devices, Sunnyvale, Calif.).

Results and Discussion—Chickens are the only species other than humansknown to generate an immune response to injected Neu5Gc (Kawachi, etal., supra; Fujii, Y., Higashi, H., Ikuta, K., Kato, S. and Naiki, M.(1982), Mol. Immunol. 19:87-94; Hirabayashi, Y., Suzuki, T., Suzuki, Y.,Taki, T., Matsumoto, M., Higashi, H. and Kato, S. (1983), J. Biochem.(Tokyo) 94:327-330; and Asaoka, H., Nishinaka, S., Wakamiya, N.,Matsuda, H. and Murata, M. (1992), Immunol. Lett. 32:91-96).Hirabayashi, et al., supra, attempted to affinity purify such antibodiesusing a Neu5Gc-containing glycolipid column, but did not rigorouslydefine specificity. The method described below is a multi-step approachto affinity chromatography that optimizes the specificity and avidity ofthe final preparation (See FIG. 1 for details). The antibody preparationthat bound to the fetuin column and then rebound to a modified fetuincolumn reacted with synthetic Neu5Gc-LacNAc-PAA but not withNeu5Ac-LacNAc-PAA, which differ by only a single oxygen atom (FIG. 2).It also recognized many proteins in bovine and chimpanzee sera, but notin human serum (data not shown). Note that chimpanzee proteins are˜99-100% identical to those of humans) (Goodman, M. (1999), Am. J. Hum.Genet. 64:31-39). Sialidase pretreatment abrogated reactivity (data notshown). Antibody specificity was confirmed for immunohistochemistryanalyses (not shown), where chimpanzee tissue sections stained intenselyand human tissues showed only a limited staining, (the latter was latershown to be specific, see below). In addition to the original immunogen(Neu5Gcα2-3Galβ1-4Glc1-1'Ceramide), this preparation recognizes Neu5Gcin either α2-3 or α2-6 linkage to underlying Galβ1-4GlcNAc(LacNAc)-PAA(not shown). This reactivity was also blocked by chimpanzee, but nothuman serum (not shown). Thus, this polyclonal preparation containsantibodies recognizing Neu5Gc on both glycoproteins and glycolipids,regardless of the linkage (α2-3 or α2-6), and regardless of theunderlying sugar chain. Binding to synthetic PAA-based probes confirmsthat the nature of the protein or the lipid tail of glycolipids are notimportant binding determinants.

Example 2 Studies Using Chicken Anti-Neu5Gc Antibody

Immunohistochemistry using the Chicken Anti-Neu5Gc Antibody—Humantissues were collected from autopsies or unused pathological material,frozen in OCT compound (VWR International) and archived at −70° C.Frozen chimpanzee tissue was provided by the Yerkes Primate Center,Atlanta, Ga. Frozen tissue sections were air-dried for 30 min., fixed in10% buffered formalin for 30 min, endogenous peroxidase activityquenched and non-specific binding sites blocked with 5% (Neu5Gc free)human serum in PBS for 30 min. Sections were then incubated with theanti-Neu5Gc antibody in 5% human serum/PBS at a 1:200 dilution at roomtemperature (RT) for 2 h. After washing, HRP-conjugated donkeyanti-chicken IgY antibody in 5% human serum/PBS at a 1:100 dilution wasapplied for 1 hr. Control sections were incubated with secondary reagentonly. Specific binding was detected using the Nova Red substrate kit(Vector Laboratories, Burlingame, Calif.), followed by hematoxylincounter-staining.

The staining pattern is summarized in Table I below: TABLE 1 Reactivityof normal human tissue sections with the anti-Neu5Gc antibody. TissueStaining Pattern* Lung Endothelium of blood vessels: Luminal edges ofbronchiolar epithelial cells and associated secretions. Skin Endotheliumof blood vessels: Luminal edges of epithelial cells of eccrine glandsand associated ducts. Colon Endothelium of blood vessels: Luminal edgesof colonic epithelial cells and associated secretions. ProstateEndothelium of blood vessels: Cytoplasmic staining of glandularepithelial cells (faint). Uterus Epithelial cells of Endometrial glands(non-secretory phase studied). Kidney Edges of collecting ductepithelium and associated secretions: No glomerular staining. SpleenEndothelium of blood vessels: Periarteriolar lymphoid sheathlymphocytes. Pancreas Endothelium of blood vessels: Epithelial cells ofacini (faint). Testis Endothelium of blood vessels: Basal cell layer(spermatogonia) of the seminiferous tubules (faint). Liver Endotheliumof blood vessels: Luminal edges of some bile duct epithelial cells.Fetal Endothelium of blood vessels: Luminal edges of epithelial Stomachcells of the villi and associated secretions. Placenta Endothelium.Breast Majority of carcinoma cells and some blood vessels. Carcinoma

Staining with the antibody showed cell-type specific reactivity in adulthuman tissues. Breast carcinomas showed reactivity in the majority oftumor cells and some related blood vessels. Reactivity was alsoconfirmed in 3/5 colon, 3/3 lung and 2/4 ovarian carcinomas and in 1/2melanomas (unpublished). In the fetus, staining was prominent inepithelial cells and secretions, as well as in the placental villusblood vessels. The overall pattern of staining in 12 normal humantissues can be summarized as prominent in secretory epithelia andassociated secretions, and present in many blood vessels. Secondaryantibody alone gave no staining (not shown). The ability to specificallyinhibit staining by chimpanzee serum or by porcine mucin varied withindividual human tissues (not shown). However, in comparison towild-type mice, no clear staining was seen in tissues from CAMH nullmice that are expected to be Neu5Gc deficient (unpublished collaborationwith Takematsu et al.) Thus, the variable inhibition by chimpanzee serumor porcine mucin in different human tissues may reflect expression ofdifferent types of Neu5Gc-containing epitopes. Of course, it could notbe ruled out that a novel human-specific epitope that is not Neu5Gc wascross-reacting with this extensively characterized antibody, so MSanalysis was performed.

Detection of Neu5Gc by HPLC Analysis and Mass Spectrometry (MS)—Humanautopsy tissues (0.5 g) were subjected to sequential organic extractionsand glycopeptides preparation (Muchmore, et al., supra). Sias inglycopeptides and lipid extracts were released by acid and purified byion exchange chromatography (Varki, A. and Diaz, S. (1984), Anal.Biochem. 137:236-247), derivatized with 1,2-diamino-4,5-methylenedioxybenzene (DMB) and analyzed by HPLC (DMB-HPLC) and MS (Chou, et al.,supra, and Klein, A., Diaz, S., Ferreira, I., Lamblin, G., Roussel, P.and Manzi, A. E. (1997), Glycobiology 7:421-432).

A Neu5Gc standard gave signals at m/z 442 and 424, consistent withmolecular ions of DMB-derivatized Neu5Gc and its dehydrated form (notshown). As expected, selective secondary MS on the m/z 442 ion peak gavethe m/z 424 dehydrated form (not shown). MS on putative-DMB-derivatizedNeu5Gc from human kidney also gave ion peaks at m/z 442 and 424 (FIG.6A). Selective secondary MS of the m/z 442 ion gave a single ion peak atm/z 424, confirming the presence of Neu5Gc (FIG. 6B). Analysis ofglycopeptides and some glycolipid fractions from human heart, liver andspleen tissues gave similar spectra. MS done in this manner is not trulyquantitative. However, there were previously reported small HPLC peaksfrom normal adult human tissues eluting in the position expected forNeu5Gc (Muchmore, et al., supra). These peaks (now confirmed to beNeu5Gc) represented<1% of total Sias in liver, skin and kidney; ˜1% inspleen and testes; and, ˜2% in the heart.

Purification of Neu5Gc from porcine submaxillary mucin—Frozen porcinesubmaxillary glands (Pel-Freeze Biologicals, Rogers, Ariz.), wereminced, homogenized in water, and total mucins precipitated from thewater-soluble supernatant by slow acidification to 100 mM acetic acid inthe cold for a few hours. The precipitate was resuspended in water, thepH neutralized, and dialyzed against water. Sia content was determinedby DMB-HPLC analysis. Sias were released with mild acid, collected bydialysis (1000 molecular-weight-cut-off), de-O-acetylated and purifiedon ion exchange columns (Gottschalk, et al., supra). Remainingcontaminants were removed with a C18 cartridge. The yield was 563 mg ofSia (95% Neu5Gc, 5% Neu5Ac) from 28.2 g of mucin.

Example 3 Uptake of Neu5Gc by Cultured Human Cells

Feeding of Human Epithelial Cells with Neu5Gc—Caco-2 cells (humanepithelial cells isolated from a primary colon carcinoma, ATCC,Manassas, Va.), were propagated in α-MEM (Gibco, Invitrogen Corporation,San Diego, Calif.) supplemented with 20% heat inactivated Fetal CalfSerum, at 37° C., 5% CO₂. Prior to feeding, the cells were split andcultured for 4 days in α-MEM supplemented with 20% heat inactivatedpremium human serum type AB (Irvine scientific, Santa Ana, Calif.).Cells were then cultured in the presence of various concentrations offree Neu5Gc (0, 0.3, 1 and 3 mM, respectively) for 3 days at 37° C.Cells were then washed with cold PBS, scraped into 20 mM sodiumphosphate pH 7.5 and sonicated. The protein extract was quantified andanalyzed by Western blot using the anti-Neu5Gc antibody.

Western Blot Analysis—Serum proteins or total proteins extracted fromNeu5Gc-fed or non-fed Caco-2 cells were either treated or sham treatedwith 10 mU Arthrobacter ureafaciens sialidase (EY Labs, San Mateo,Calif.) in 100 mM sodium acetate, pH 5.5 at 37° C. for 3 h. AfterSDS-PAGE electrophoresis, the separated proteins were transferred tonitrocellulose membrane, and binding of the anti-Neu5Gc antibody(1:10,000 in Tris-buffered saline with 0.1% Tween 20 (TBST)) wasdetected using a secondary HRP-conjugated donkey anti-chicken IgYantibody (1:30,000 in TBST) and Supersignal West Pico (Pierce, Rockford,Ill.).

Although human cells cultured in fetal calf serum express cell surfaceNeu5Gc in small amounts (Muchmore, et al., supra, and Oetke, et al.,supra), this could represent either metabolic incorporation ornon-specific absorption. Human epithelial cells were cultured in humanserum until no traces of Neu5Gc derived from fetal calf serum could bedetected by HPLC (typically ˜4 days). Culture was then continued for 3days in the absence or presence of varying amounts of free Neu5Gc. HPLCanalysis showed increasing incorporation of Neu5Gc into the cells overtime, with the highest level reaching 85% of total Sia, after incubationin 3 mM Neu5Gc for 3 days (data not shown). Metabolic incorporation ofNeu5Gc into glycoproteins was confirmed by Western blotting using theanti-Neu5Gc antibody (data not shown). Staining was only seen whenNeu5Gc was present in the medium, and reactivity was eliminated bysialidase treatment (further confirming the specificity of theantibody). These data suggested that the small amounts of Neu5Gc innormal human tissues could originate from dietary sources.

Example 4 Human Ingestion Studies

Human Neu5Gc Ingestion Study—Human subjects were enlisted in the study,and were told to avoid animal foods for 2 days, all medications for 1day, use a lanolin-free (Neu5Gc-free) shampoo for 2 days, and ingestonly fruit juice on the morning of study. Subjects then drank 150 mg ofporcine submaxillary mucin Sias (see above) dissolved in 100 ml water.For the next 6 h they drank a 1:1 mixture of fruit juice and soymilk(Sia free) at ˜2 ml/Kg/hr. Serum, urine and saliva samples were obtainedat multiple time points. Human saliva was collected by chewing parafilm,after washing the mouth out well with water. Urine volume and pH wererecorded prior to centrifugation at 1000 g for 10 min, and collection ofthe supernatant. All samples were stored frozen at −20° C. Head hairs(˜20) and/or facial hair trimmings (from men) were collected prior to,and on several days after Neu5Gc ingestion.

Purification and analysis of Sialic Acids and N-acylmannosamines fromHuman Samples—Human salivary mucin was precipitated by adjusting to pH 3with 100 mM acetic acid, stirring at 4° C. overnight, collected at14,000 g for 10 min, and washed twice with 1 ml of ice-cold PBS. Humanhair clippings were washed in 100 mM ice-cold acetic acid for two h andcrushed in a clean ceramic mortar. Bound Sias from saliva, hair or serumwere released with mild acid as above, filtered through Microcon 10filters (Millipore, Bedford, Mass.), dried down, reconstituted in waterand analyzed for Sia content by DMB-HPLC.

Five ml urine aliquots were diluted 5-fold and loaded onto 10 ml columnsof AG50W-X2 resin (H⁺ form, BioRad, Richmond, Calif.) equilibrated inwater. The run-through and washes (2×10 ml of water) were pooled andloaded onto 10 ml columns of AG1X8 resin (Formate form, BioRad)equilibrated in water. The combined run-through and washes (2×10 ml ofwater) were lyophilized. After washing with 5 volumes of 10 mM formicacid, free Sias were eluted with 5 volumes of 1M formic acid, dilutedtwo-fold with water, and lyophilized.

The neutral fraction from urine was dissolved in water and reducedperacetylated volatile derivatives of free sugars prepared (Bjorndal,H., Lindberg, B., Pilotti, A. and Svensson, S. (1967), Carbohydr. Res.5: 433-440). Samples were dissolved in 10 ul of acetone, and gas-liquidchromatography for N-acylmannosamines performed on 1 ul aliquots(Bjorndal, et al., supra). Eluting ions were detected and fragmentedusing a HP5971 Mass Selective Detector (Hewlett-Packard, Palo Alto,Calif.).

Free Sias were purified from urine samples, and analyzed for Neu5Gccontent by DMB-HPLC. Since overlapping fluorescent peaks preventeddefinitive identification and quantification, HPLC fractions eluting atthe time expected for DMB-Neu5Gc were collected and subjected to MS,confirming an increase in ion peaks at m/z 424 and m/z 442. The sum ofion intensities of m/z 424 and m/z 442 at each time point provided anestimate of Neu5Gc quantity. Ingested Neu5Gc was rapidly absorbed andexcreted into the urine (FIG. 3A). About 3-6% of the ingested dose wasexcreted within 4-6 h, with the peak excretion rate at 2-3 h and areturn to baseline levels within 24 h. Notably, small amounts of Neu5Gcwere also present at baseline.

Sias could be converted into their corresponding acylmannosamines bysialate:pyruvate-lyases (Schauer, —R., Sommer, U., Krüger, D., Van, U.H. and Traving, C. (1999), Biosci. Rep. 19:373-383), derived from eitherintestinal cells or gut microbes. Such molecules could then enter cellsand be reconverted back to the original Sias (Kayser, H., Zeitler, R.,Kannicht, C., Grunow, D., Nuck, R. and Reutter, W. (1992), J. Biol.Chem. 267:16934-16938). To ascertain potential conversion of theingested Neu5Gc into N-glycolylmannosamine (ManNGc), presence of thisneutral sugar in the urine was investigated. Small amounts of ManNGcwere present at baseline in one subject (not shown). There was noobvious increase over time in any subject (not shown), indicating thatthe remaining ingested Neu5Gc was not absorbed and excreted as ManNGc.Thus, much of the uptake is apparently occurring as free Neu5Gc.

The possibility that ingested Neu5Gc was incorporated into newlysynthesized glycoproteins was also investigated. Mucins from allbaseline saliva samples showed very small amounts of Neu5Gc detectableby MS. An increase 2 days after ingestion occurred in 2 of 3 subjects,with a decline to baseline levels by 4 days (FIG. 3B). While Neu5Gc wasnot detected in serum samples at any time points, small amounts werefound in facial hair clippings at baseline for two subjects, withincreases after ingestion. Overall the data show uptake and excretion ofintact Neu5Gc, and low-level metabolic incorporation into newlysynthesized glycoconjugates. Although there were no immediate reactionsto Neu5Gc ingestion, this does not rule out long-term consequences.

Example 5 Anti-Sia Antibodies in Human Sera

Detection of Anti-Neu5Gc and anti-α-Gal specific antibodies in humansera—Microtiter (Costar, Corning, N.Y.) plate wells were coated withNeu5Ac-polyacrylamide(PAA), Neu5Gc-PAA, or Galα1-3GalBeta1-4GlcNAc-PAA(Glycotech, Rockville, Md.) at a concentration of 500 ng/well, in 50 mMsodium carbonate-bicarbonate buffer pH 9.5, at 4° C. overnight. Afterwashing with TBS, pH 7.5 and blocking with TBST for 2 h at RT,triplicate wells were incubated with 1:50 dilutions of serum in TBST atRT for 4 h. Wells were washed 5 times with TBS, and incubated withHRP-conjugated mouse anti-human IgA (Calbiochem, San Diego, Calif.),anti-human IgG (Jackson ImmunoResearch, West Grove, Pa.) or anti-humanIgM (KPL, Gaithersburg, Md.) each diluted in TBST at 1:20,000, at RT for1.5 hour. The IgG anti-Neu5Gc and anti-α-Gal antibodies were quantitatedusing a standard curve of normal human IgG coated to the wells under thesame general conditions.

Anti-Neu5Gc antibodies of the IgA, IgM and IgG classes were detected inthe sera of all three subjects who ingested Neu5Gc at baseline, but noincreases occurred after Neu5Gc ingestion (not shown). Studies ofadditional normal adults showed that 16/18, 14/18 and 17/18 sera werepositive for IgA, IgM and IgG antibodies, respectively (FIG. 4A).Specificity was confirmed by showing that anti-Neu5Gc reactivity wasblocked by chimpanzee serum in a concentration-dependent manner, but notby equivalent amounts of a Neu5Gc antibody-negative human serum (FIG.4B). The chimpanzee serum used for inhibition was also negative foranti-Neu5Gc antibodies (FIG. 5). Further confirmation came from >70%inhibition of reactivity by free Neu5Gc but not by Neu5Ac (each at 2 mM,not shown).

Some human sera required up to 3-4% chimpanzee serum to obtain completeblockade (FIG. 4B). Since chimpanzee serum glycoproteins carry ˜0.1 nmolof Neu5Gc per ul, the anti-Neu5Gc antibodies must be present atrelatively high concentrations in such human sera. Indeed, theseantibodies represented up to 0.25% of the total circulating IgG in somesubjects (FIG. 5). This falls into the range of the well-knownxeno-reactive antibodies against the non-human α-Gal epitope (Galili, U.Immunol Today 14:480-482; and Joziasse, D. H. and Oriol, R. (1999),Biochem. Biophys. Acta Mol. Basis. Dis. 1455:403-418). We also comparedlevels of IgG anti-Neu5Gc and anti-α-Gal antibodies. As expected fromprior literature, all the human sera contained easily detectableanti-α-Gal antibodies. Anti-Neu5Gc levels were highly variable, andthere was no direct correlation between the two antibody levels in anygiven individual. On the other hand, the relative levels of anti-Neu5Gcantibodies between individuals were reproducible on repeat sampling.Four chimpanzee sera showed no anti-Neu5Gc antibodies, but clearevidence of anti-α-Gal antibodies (FIG. 5).

Example 6 Sialic Acids in Food Samples

Purification and Analysis of Sialic Acids from Food Samples—One gramportions of samples were homogenized in 2M acetic acid, and total Siasreleased at 80° C. for 3 h. After clarification at 50,000 g, thesupernatant was filtered through a Microcon 10 unit, dried down,reconstituted in water, de-O-acetylated with 0.1M NaOH for 30 min at 37°C., and neutralized with HCl. Aliquots were derivatized with DMB andNeu5Gc content analyzed by HPLC and MS as described above.

The results are shown below in Table 2: TABLE 2 Preliminary survey oftotal sialic acid and Neu5Gc content of common food items Total wtNeu5Gc Neu5Gc Total Neu5Gc if eaten intake if per % Sia,*, content,Serving Servings daily, eaten Serving, Food item Neu5Gc* μg/g μg/gsize,^(†) g per day† g/day daily,^(‡) μg μg Cod <0.1 40 <0.04 90 3 270<11 — Salmon <0.1-3.5 49 1.47 90 3 270 <11-397 132 Tuna <0.1 32 <0.03290 3 270 <9 — Chicken <0.1 76 <0.076 90 3 270 <21 — Turkey <0.1-0.6 46<0.046 90 3 270 <12-75  — Duck <0.1 20 <0.02 90 3 270 <5 — Cow milk (2%)3 258 7.74 237 2 474 3,668 1,834 Cow milk (raw) 3 262 7.86 237 2 4743,726 1,863 Butter 3 40 1.2 5 3 15 18 6 Cow cheese 4 160 6.4 50 3 150960 320 Goat cheese 42 95 39.9 44 3 132 5,267 1,756 Lamb 18 101 18.2 903 270 4,914 1,638 Pork 19 134 25.5 90 3 270 6,885 2,295 Beef^(§)  31-4370 30.1 90 3 270 5,859-8,127 2,709*Total Sia content and percent of Neu5Gc in each food sample wasquantified as described herein. Range of values obtained are shown forfood items that were studied more than once. Oral intake calculationsinclude the highest values in such cases.^(†)Recommended daily servings and serving sizes of each item are basedon guidelines set by the U.S. Department of Agriculture [U.S. Departmentof Agriculture (2000) Nutrition and Your Health: Dietary Guidelines forAmericans (U.S. Department of Agriculture, Washington, DC), Home GardenBulletin 323, 5th Ed.].^(‡)Oral intake calculation only. Actual bioavailability is unknown.^(§)Lean beef and beef fat portions had similar Neu5Gc contents (datanot shown).

As shown above, Neu5Gc is enriched in food sources of mammalian origin.Sias have never been detected in plants, and are found in large amountsprimarily in vertebrates and in a few “higher” invertebrates (Rosenberg,et al., supra; Schauer, et al., supra; and Warren, L. (1963), Comp.Biochem. Physiol. 10:153-1712). While several pathogenic microbes canexpress Neu5Gc, none have been reported to express Neu5Gc.

As shown above in Table 2, the highest amount was found in lamb, porkand beef (so-called “red meat”). In contrast, levels were very low orundetectable in poultry and in fish. Hen's eggs contain primarily Neu5Ac(Juneja, L. R., Koketsu, M., Nishimoto, K., Kim, M., Yamamoto, T. andItoh, T. (1991), Carbohydr. Res. 214:179-186). Intermediate amounts werefound in bovine milk and milk products, with higher levels in goat andsheep milk products. As summarized in Table 2, USDA recommended dietaryintake would result in substantial intake of Neu5Gc from red meats, andmoderate amounts from dairy products. This survey is a limited samplingof human food. A much broader survey using the same techniques is neededto fully establish the possible sources of Neu5Gc in human diet.

Example 7 Neu5Gc Metabolism

Using the methods described herein, Neu5Gc is detectable in fetal smallintestine and placenta, and its presence in 9/9 breast 3/5 colon, 3/3lung and 2/4 ovarian carcinomas and in 1/2 melanomas has been found.Surprisingly, secretory epithelia and endothelium within certain normaladult human tissues (lung, skin, colon, prostate, uterus, testis) alsoshowed reactivity with anti-Neu5Gc antibodies. The presence of Neu5Gc insuch samples was confirmed by HPLC and MS. (Tangvoranuntakul, P.,Gagneux, P., Diaz, S., Bardor, M., Varki, N., Varki, A. & Muchmore, E.(2003). Proc Natl Acad Sci USA 100:12045-12050.)

Cultured Human cells can also take up and incorporate free Neu5Gc orNeu5Gc from fetal calf serum glycoproteins. The hydroxylase activityresponsible for generating Neu5Gc is encoded by the single copy CAMHgene, which has been highly conserved from Sea Urchins to Primates. Theonly known homologs of this gene are distantly related hydroxylases inbacteria and plants (Schmidt, C. L. and Shaw, L. A (2001). J. Bioenerg.Biomembr. 33:9-26. An alternative pathway for Neu5Gc synthesis involvingGlycolyl-CoA as a donor has been postulated only in principle, but neverproven (Vamecq, J. & Poupaert, J. H. (1990). Biochem. Cell Biol.68:846-851; and Vamecq, J., Mestdagh, N., Henichart, J.-P. and Poupaert,J. (1992). J Biochem (Tokyo) 111:579-583.

It is therefore reasoned that the Neu5Gc in normal humans originatesfrom exogenous sources. Indeed, human cells fed with free Neu5Gcincorporated it into endogenous glycoproteins, achieving levels of >50%,comparable to those found in ape cells. Likewise, human cells culturedin FCS incorporated Neu5Gc from the serum glycoproteins into the nativeglycoconjugates of the cells (Muchmore, E. A., Diaz, S. and Varki, A.(1998). Am J Phys Anthropol 107:187-198; and Tangvoranuntakul, et al.,supra.

It was also found that the uptake of both free Neu5Gc andNeu5Gc-containing proteins involves micro- and macro-pinocytosis,delivery to the lysosome, and export into the cytosol by the lysosomalSia transporter (in the case of serum protein-bound Neu5Gc, release by alysosomal sialidase is required before transport into the cytosol) (FIG.7). Once free Neu5Gc has reached the cytosol, it is available foractivation to CMP-Neu5Gc and eventual transfer to glycoproteins, in amanner similar to endogenously synthesized Neu5Gc. Since Neu5Gc waseliminated during human evolution only ˜3 million years ago, it is notsurprising that the enzymes and transporters involved in activation,transport and transfer of Sias do not see it as “foreign”. (Chou, H. H.,et al., supra.)

It is also suspected that the lysosomal sialic acid transporter iscritically involved in the uptake of Neu5Gc. To study transport, normaland lysosomal Sia transporter mutant human fibroblasts (GM 08496 and GM05520) were fed for 3 days with 3 mM Neu5Gc, ManNGc or Neu5Ac. The cellswere then harvested and fractionated and the Sia content in thedifferent fractions analysed by DMB derivatization followed by HPLC. The% of Neu5Gc incorporation was reduced from 37% in normal fibroblasts to5% in the mutant cells. As a control, the incorporation of ManNGc intoNeu5Gc was also studied, which presumably occurs via passive diffusionthrough the plasma membrane, and does not require the lysosomaltransporter. Indeed, it was found that there was essentially nodifference between normal (19% Neu5Gc) versus mutant fibroblasts (18%Neu5Gc) following incubation with ManNGc. To further confirm that therewas a difference in incorporation, a Western blot analysis of proteinsextracted from the membrane of wild-type and GM 05520 mutant humanfibroblasts using an anti-Neu5Gc antibody, with or without prior Neu5Gcor ManNGc feeding. This confirmed that the mutant fibroblasts could notincorporate Neu5Gc into glycoproteins, but could in fact convert it fromManNGc. Taken together, the data confirms the hypothesis that thelysosomal sialic acid transporter plays a crucial role in deliveringfree sialic acids that enter the cell via endocytosis to the cytosol foractivation and incorporation as depicted in FIG. 7.

Example 8 Neu5Gc Antibody-Mediated Cell Destruction

Naturally occurring human antibodies (Nabs) against Neu5Gc (anti-Neu5Gc)can cause complement deposition and/or cell death of human cells loadedwith Neu5Gc. To explore the possibility that the human anti-Neu5Gc Nabscan cause inflammation and/or cell killing, human cells were cultured inhuman serum, with or without free Neu5Gc in the medium. It has beenfound that upon exposure to human sera known to contain anti-Neu5GcNAbs, the Neu5Gc-loaded cells showed reduced viability, deposition ofcomplement (C3b) and cell killing (data not shown). There was also acorrelation of extent of cytotoxicity with the relative amounts of NAbs.Cytotoxicity could be blocked by small amounts of chimpanzee serum,which is rich in Neu5Gc, but is otherwise>99% identical to human serum,which does not block this killing (data not shown). Accordingly, it islikely that anti-Neu5Gc Nabs antibodies may have an undesirable affecton cells in vivo.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A method of determining % N-glycolylneuraminic acid (Neu5Gc) of abiological material comprising the steps of: (a) measuring the amount ofN-acetylneuraminic acid (Neu5Ac) present in the material per givenweight; (b) measuring the amount of Neu5Gc present in the material pergiven weight; and (c) calculating the % Neu5Gc of the sample, wherein %Neu5Gc is determined using the formula: %Neu5Gc=[(Neu5Gc)/(Neu5Ac+Neu5Gc)]×100.
 2. The method of claim 1, whereinthe biological material is a food sample.
 3. The method of claim 2,wherein the food sample is a red meat or a dairy product.
 4. The methodof claim 2, further comprising the step of calculating the Neu5Gccontent per serving by multiplying the amount of Neu5Gc in step (b) by afood serving size of a given weight.
 5. The method of claim 1, whereinthe biological material is a clinical sample.
 6. The method of claim 5,wherein the clinical sample is from an animal source.
 7. The method ofclaim 6, wherein the clinical sample is urine, tissue, blood or saliva.8. The method of claim 5, wherein the tissue is suspected of beingdiseased.
 9. The method of claim 5 further comprising the step ofrepeating steps (a) to (c) at more than one time period.
 10. The methodof claim 9, wherein the clinical sample is from a human or non-humananimal subject, and wherein the subject undergoes a dietary change ortherapy during the time period.
 11. A method of purifying sialicacid-specific antibodies, comprising the steps of: (a) preparingantibodies to sialic acids; (b) contacting the antibodies from step (a)with a first solid phase to which a sialic acid having a side chain hasbeen attached; and (c) contacting the antibodies that bound to the firstsolid phase with a second solid phase to which the sialic acid withoutthe side chain has been attached.
 12. The method of claim 11, whereinthe sialic acid having a side chain is selected from the groupconsisting of: N-acetylneuraminic acid, Ketodeoxynonulosonate,N-glycolylneuraminic acid, N-propanoylneuraminic acid,N-butanoylneuraminic acid, N-pentanoylneuraminic acid,N-hexanoylneuraminic acid, N-heptanoylneuraminic acid,N-oxooctanoylneuraminic acid, N-levulinolylneuraminic acid,N-homolevulinoylneuraminic acid, N-oxohexanoylneuraminic acid,N-oxoheptanoylneuraminic acid, and N-oxooctanoylneuraminic acid.
 13. Themethod of claim 11, wherein the side chain is removed using periodate.14. A method of detecting anti-Neu5Gc specific antibodies in abiological material comprising the steps of: (a) determining the amountof anti-Neu5Ac antibodies present in the material using Neu5Gc as anantigen target; (b) determining the amount of anti-Neu5Gc antibodiespresent in the material using anti Neu5Gc as an antigen target; and (c)subtracting the amount of anti-Neu5Ac antibodies from the amount ofanti-Neu5Gc antibodies to determine the amount of anti-Neu5Gc specificantibodies.
 15. The method of claim 14, wherein step (a) and step (b)are performed in identical systems, except that in step (a), Neu5Ac isthe antigen and in step (b), Neu5Gc is the antigen target.
 16. Themethod of claim 15, wherein the biological material is a clinicalsample.
 17. The method of claim 16, wherein the clinical sample is froma human subject.
 18. The method of claim 17, wherein the clinical sampleis blood.
 19. A composition comprising an affinity purified antibodyspecific for binding N-glycolylneuraminic acid (Neu5Gc).
 20. A methodfor purification of antibodies specific to sialic acid groupscomprising: (a) exposing an antibody containing solution to a firstimmobilized target that has a very low density of the sialic acid underconditions to allow antibody binding; (b) challenging the boundantibodies with a second immobilized phase comprising the firstimmobilized phase treated with mild sodium periodate; and collecting theantibodies that do not bind to the second immobilized phase.
 21. Amethod for the diagnosis of disease comprising: (a) collecting a serumor tissue sample from an individual; (b) analyzing the sample for anelevated level of Neu5Gc specific antibodies in serum; and (c)correlating an increased level of antibody with the presence of disease.